exoEasy Maxi Kit

For purification of exosomes and other extracellular vesicles (EVs) from plasma, serum and cell culture supernatant

Isolate intact exosomes and other extracellular vesicles

Suitable for functional testing and physical and biochemical analysis

Purify exosomes and other extracellular vesicles in 25 minutes

Process multiple samples in parallel using a simple spin-column procedure

Use with up to 4 ml of plasma or serum or 32 ml of cell culture supernatant

The exoEasy Maxi Kit uses membrane affinity spin columns to efficiently isolate exosomes and other extracellular vesicles from serum, plasma, cell culture supernatant and other biological fluids. The maxi column format allows the use of large sample volumes, including up to 4 ml plasma or serum or up to 32 ml cell culture supernatant. The procedure is very fast, consistent and highly suited for isolation of EVs for functional analysis of exosomes and other extracellular vesicles.

The exoEasy Maxi Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Intact vesicles are eluted from the exoEasy membrane with higher purity compared with ultracentrifugation.

Scanning electron microscopy (SEM; 20,000x magnification) was performed on a solubilized pellet from ultracentrifugation of pre-filtered (0.8 µm) plasma compared to an exoEasy eluate. Both preparations contain vesicle-shaped structures with an expected size range from 50–200 nm (white arrows; scale bar 200 nm). The preparation from ultracentrifugation also includes many smaller, unidentified structures that do not match the expected shape and size of extracellular vesicles.

The exoEasy Maxi Kit – exosome purification in just 25 minutes.

The exoEasy Maxi Kit allows isolation of extracellular vesicle from pre-filtered serum, plasma or cell culture supernatant in just 25 minutes. Eluted vesicles are ready for physical or biochemical characterization, or buffer exchange and concentration using e.g., ultrafiltration.

(A) HeLa cells were maintained in serum-free medium for 48 h before harvesting culture supernatants. The exoEasy Maxi Kit was used to isolate EVs from 2 ml of supernatant that had been pre-filtered using a 0.8 µm filter. EVs were diluted 1:100 and characterized using the Nanosight NS300 instrument (Malvern, UK). (B) EVs were also isolated from 1 ml of EDTA plasma (measured at 1:200). Mean particle diameters were reported as 212 nm and 214 nm, respectively, which is within the expected range for a mixed population of exosomes and other extracellular vesicles. Red outline indicates variation between 5 repeat measurements.

Extracellular vesicles isolated using the exoEasy Kit are taken up efficiently by target cells (customer data).

HEK 293T cells were grown in vesicle-free DMEM for 66 h. Extracellular vesicles (EVs) were isolated using the exoEasy Kit from 15 ml cell culture supernatant pre-filtered using a 0.8 µm filter. Particle concentration was determined using the Nanosight instrument. Isolated EVs were labeled using BODIPY TR Ceramide for 20 min at 37°C. Unincorporated dye was removed by size exclusion chromatography (SEC). As a negative control, an equivalent amount of free dye in PBS was subjected to the same SEC cleanup. To test the uptake of EVs into target cells, approximately 1.5 x 109 labeled particles (representing about 1% of the total eluate from an exoEasy preparation started with 15 ml medium) were incubated with 2 x 104 HEK 293T cells in 200 µl DMEM for 1 h at 37°C. After washing, cells were stained with DAPI to visualize nuclei (bottom row) versus EVs taken up by cells (top row). Left column: negative control, right column: labeled EVs.

Performance

The exoEasy protocol for purification of exosomes and other extracellular vesicles is not only faster than ultracentrifugation, but also yields a cleaner preparation. Scanning electron microscopy shows that while both techniques deliver intact vesicles of the expected size, the preparation from ultracentrifugation also contains many smaller structures that do not match the expected size or shape for EVs (see figure “Intact vesicles are eluted from the exoEasy membrane with higher purity compared with ultracentrifugation”).
Isolated vesicles are fully functional and can be used for several downstream applications including cell culture work (see figure “Extracellular vesicles isolated using the exoEasy Kit are taken up efficiently by target cells (customer data)”). Quality and quantity of eluted vesicles can be determined by a range of different methods. These include electron microscopy, physical characterization (e.g., Nanoparticle Tracking Analysis (NTA), or Tunable Resistive Pulse Sensing (TRPS)) and analysis of molecular constituents of vesicles, such as proteins, nucleic acids or lipids. Since particle counting techniques like NTA or TRPS do not distinguish vesicles from other particulate matter, particle counts performed in unprocessed biological fluids or crude preparations that still contain other particulate matter (e.g., protein complexes) may vastly overestimate the number of vesicles present.

The exoEasy Maxi Kit uses a membrane-based affinity binding step to isolate exosomes and other EVs from serum and plasma or cell culture supernatant. The method does not distinguish EVs by size or cellular origin, and is not dependent on the presence of a particular epitope. Instead, it makes use of a generic, biochemical feature of vesicles to recover the entire spectrum of extracellular vesicles present in a sample (see figure “Characterization of extracellular vesicles isolated using the exoEasy Maxi Kit by
NTA”). It is therefore essential to completely remove cells, cell fragments, apoptotic bodies, etc., by centrifugation or filtration of samples before starting the protocol.

The technology developed with Exosome Diagnostics, Inc., uses a spin column format and specialized buffers to purify exosomes from pre-filtered biological fluids; up to 4 ml using plasma or serum. For cell culture supernatants, processing of up to 32 ml sample (4 column loading steps) has been successfully tested. However, the concentration of vesicles in supernatants depends strongly on the cell type and culture conditions; therefore, we recommend starting with no more than 16 ml of supernatant for sample material that has not been tested with the kit previously. Higher sample volumes may result in reduced recovery of vesicles. The procedure is very fast, consistent and highly suited for functional analysis of exosomes and other extracellular vesicles. Particulate matter other than vesicles, such as larger protein complexes that are especially abundant in plasma and serum, is largely removed during the binding and wash steps.

Procedure

The total procedure for isolating extracellular vesicles takes just 25 minutes. Pre-filtered plasma, serum or cell culture supernatant (with particles larger than 0.8 μm excluded) are mixed with Buffer XBP and bound to an exoEasy membrane affinity spin column. The bound exosomes are washed with Buffer XWP, eluted with 400 µl Buffer XE (an aqueous buffer containing primarily inorganic salts) and are then ready to use in physical or biochemical analysis. For certain applications, such as biological assays, an additional concentrating or buffer exchange step may be required. This can be achieved using ultrafiltration with a pore size of 100 kDa or smaller.

Applications

The exoEasy Kit is highly suited for isolating microvesicles found in serum, plasma, cell culture supernatant and other biological fluids. Isolated vesicles can be analyzed by and used for:

Physical characterization (e.g., NTA, TRPS)

Electron microscopy

Isolation and analysis of DNA, RNA*, proteins, lipids and other constituents